Endothelins are peptides that exist in three isoforms, ET-1, ET-2, and ET-3, each encoded by a distinct gene. They have been originally discovered in the conditioned medium of porcine endothelial cells in 1988 by Yanagisawa (Yanagisawa M; Kurihara H; Kimura S; Tomobe Y; Kobayashi M; Mitsui Y; Yazaki Y; Goto K; Masaki T: A novel potent vasoconstrictor peptide produced by vascular endothelial cells [see comments]. NATURE (1988 Mar. 31), 332(6163), 411-5.). The active ETs are peptides of 21 amino acids with two intramolecular disulfide bridges. They are produced from preproproteins of 203 to 212 amino acids, which are processed by furin-like endopeptidases to the biologically inactive big-endothelin (big-ET). The big-ETs are specifically processed to mature ETs by a hydrolytic cleavage between amino acids 21 and 22 that are Trp21-Val22 (big-ET-1, big ET-2) and Trp21-Ile22 in big-ET-3 respectively. Already in 1988 a specific metalloprotease was postulated to be responsible for this specific cleavage. In 1994 ECE-1 (endothelin converting enzyme-1) was purified and cloned from bovine adrenal (Xu D, Emoto N, Giaid A, Slaughter C, Kaw S, de Witt D, Yanagisawa M: ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell (1994) 78: 473-485).
ECE-1 is a membrane bound type II zinc-endopeptidase with a neutral pH optimum and a zinc binding motif HExxHx( greater than 20)E. It belongs to subfamily M13 and has a large 681 amino acid ectodomain that comprises the active site. Other members of the M13 family are NEP24.11 (neutral endopeptidase), PEX, a phosphate regulating neutral endopeptidase, and Kell blood group protein that has recently been described as a big-ET-3 processing enzyme. Members of the M13 family of human origin are characterized by a high molecular weight ( greater than 80 kDa) a number of conserved disulfide bridges and a complex glycosylation pattern. The structure of NEP has recently been solved. (Oefner et al, J. Mol. Biol. 2000, 296, 341-349). The catalytic domain of ECE and related human M13 proteinases are significantly larger ( greater than 650 amino acids) than members of matrix metalloproteases (MMPs). Unlike the family of the MMPs which belong to the metzincins and display a typical HExxHxxGxxH pattern members of the M13 family are gluzincins comprising a HExxHx( greater than 20)E pattern. These two families are clearly different in size of catalytic domains, structure and zinc coordinating pattern of ligands. Active sites of the two families show clear differences which has clear impact on type of inhibitors and the potential selectivity.
The present invention relates to compounds of formula (I) 
wherein
R1 is hydrogen, alkylcarbonyl, or arylcarbonyl;
R2 is alkyl, alkenyl, alkinyl, cyanoalkyl, hydroxyalkyl, carboxyalkyl, alkoxycarbonyl, alkylcarbonylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylsulfonyl, aryl, arylalkyl, arylalkoxyalkyl, aryl(alkoxycarbonyl)alkyl, arylcarbamoyl, diarylalkyl, aryl(carboxyalkyl)amide, arylamino, arylcarbonyl, arylsulfonyl, cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl, or the group YR2 is heterocyclyl or R2 is a group of the formula 
R3 is alkyl, alkylcycloalkyl, alkylcycloalkylalkyl, cycloalkyl, halogenalkyl, carboxyalkyl, aminoalkyl, dialkylaminoalkyl, alkoxyalkyl, alkoxycarbonylalkyl, alkinyl, aryl, arylalkyl, arylalkyl(alkoxycarbonyl)alkyl, arylcarbonylalkyl, aryloxyalkyl, arylalkenyl, aryl(alkoxycarbonyl)alkyl, heteroaryl, heteroarylalkyl, heterocyclyl or heterocycylalkyl, and R3 is hydroxy in case of X is SO2;
R4 is hydrogen in case m=1 or alkyl or hydrogen in case m=0;
R5 is hydrogen, alkyl, aryl, or carboxyalkyl;
R6 is hydrogen, alkyl, aryl, carboxyalkyl, arylcarbonyl, alkylcarbonyl, arylalkoxycarbonyl, or arylalkyl;
R7 is hydrogen, aryl, alkyl, arylalkyl, heterocyclylalkyl, arylamino, alkyl(arylalkyl)amino, alkoxycarbonylalkyl, carboxyalkyl, or alkylthioalkyl;
R8 is hydroxy, alkyl, aryl, cyanoalkyl, alkoxy, arylalkyl, arylalkoxy, mono- or dialkylamino, arylamino, aryl(alkyl)amino, cyanoalkylamino, arylalkyl(alkyl)amino, heteroaryl, heteroarylalkyl, or heterocyclyl; and
X is xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94NHxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)NR5xe2x80x94, C(O)Oxe2x80x94;
Y is xe2x80x94CH2, xe2x80x94Oxe2x80x94, xe2x80x94NR6xe2x80x94 or xe2x80x94Sxe2x80x94;
m and p independently are 0 or 1, n and q independently are 1, 2 or 3 and o is 0, 1 or 2 with the proviso that the sum of n, o and p is xe2x89xa72 and xe2x89xa63; and
dimeric forms, and/or pharmaceutically acceptable esters, and/or pharmaceutically acceptable salts thereof, preferably pharmaceutically acceptable esters, and/or pharmaceutically acceptable salts thereof, and most preferably pharmaceutically acceptable salts thereof.
The present invention is directed to compounds which are useful as inhibitors of metalloproteases, e.g. zinc proteases, particularly zinc hydrolases, and which are effective in the prophylaxis and treatment of disease states which are associated with vasoconstriction of increasing occurrences. Examples of such disorders are high blood pressure, coronary disorders, cardiac insufficiency, renal and myocardial ischaemia, renal insufficiency, dialysis, cerebral ischaemia, cardiac infarct, migraine, subarachnoid haemorrhage, Raynaud syndrome and pulmonary high pressure. In addition the compounds are useful as cytostatic and cerebroprotective agents for inhibition of graft rejection, for organ protection and for treatment of ophthalmological diseases.
The present invention is directed to compounds of formula (I): 
wherein
R1 is hydrogen, alkylcarbonyl, or arylcarbonyl;
R2 is alkyl, alkenyl, alkinyl, cyanoalkyl, hydroxyalkyl, carboxyalkyl, alkoxycarbonyl, alkylcarbonylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, alkylsulfonyl, aryl, arylalkyl, arylalkoxyalkyl, aryl(alkoxycarbonyl)alkyl, arylcarbamoyl, diarylalkyl, aryl(carboxyalkyl)amide, arylamino, arylcarbonyl, arylsulfonyl, cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl, or the group YR2 is heterocyclyl or R2 is a group of the formula 
R3 is alkyl, alkylcycloalkyl, alkylcycloalkylalkyl, cycloalkyl, halogenalkyl, carboxyalkyl, aminoalkyl, dialkylaminoalkyl, alkoxyalkyl, alkoxycarbonylalkyl, alkinyl, aryl, arylalkyl, arylalkyl(alkoxycarbonyl)alkyl, arylcarbonylalkyl, aryloxyalkyl, arylalkenyl, aryl(alkoxycarbonyl)alkyl, heteroaryl, heteroarylalkyl, heterocyclyl or heterocycylalkyl, and R3 is hydroxy in case of X is SO2;
R4 is hydrogen in case m=1 or alkyl or hydrogen in case m=0;
R5 is hydrogen, alkyl, aryl, or carboxyalkyl;
R6 is hydrogen, alkyl, aryl, carboxyalkyl, arylcarbonyl, alkylcarbonyl, arylalkoxycarbonyl, or arylalkyl;
R7 is hydrogen, aryl, alkyl, arylalkyl, heterocyclylalkyl, arylamino, alkyl(arylalkyl)amino, alkoxycarbonylalkyl, carboxyalkyl, or alkylthioalkyl;
R8 is hydroxy, alkyl, aryl, cyanoalkyl, alkoxy, arylalkyl, arylalkoxy, mono- or dialkylamino, arylamino, aryl(alkyl)amino, cyanoalkylamino, arylalkyl(alkyl)amino, heteroaryl, heteroarylalkyl, or heterocyclyl;
X is xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94NHxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)NR5xe2x80x94, C(O)Oxe2x80x94;
Y is xe2x80x94CH2, xe2x80x94Oxe2x80x94, xe2x80x94NR6xe2x80x94 or xe2x80x94Sxe2x80x94;
m and p independently are 0 or 1, n and q independently are 1, 2 or 3 and o is 0, 1 or 2 with the proviso that the sum of n, o and p is xe2x89xa72 and xe2x89xa63; and
dimeric forms, and/or pharmaceutically acceptable esters, and/or pharmaceutically acceptable salts thereof.
The term xe2x80x9calkylxe2x80x9d, alone or in combination, means a straight-chain or branched-chain alkyl group containing a maximum of 7, preferably a maximum of 4, carbon atoms, e.g., methyl, ethyl, n-propyl, 2-methylpropyl (iso-butyl), 1-methylethyl (iso-propyl), n-butyl, and 1,1-dimethylethyl (t-butyl).
The term xe2x80x9ccarboxyxe2x80x9d refers to the group xe2x80x94C(O)OH.
The term xe2x80x9ccarbamoylxe2x80x9d refers to the group xe2x80x94C(O)NH2.
The term xe2x80x9ccarbonylxe2x80x9d refers to the group xe2x80x94C(O)xe2x80x94.
The term xe2x80x9chalogenxe2x80x9d refers to the group fluoro, bromo, chloro and iodo.
The term xe2x80x9csulfonylxe2x80x9d refers to the group xe2x80x94S(O2)xe2x80x94.
The term xe2x80x9calkenylxe2x80x9d refers to a hydrocarbon chain as defined for alkyl having at least one olefinic double bond (including for example, vinyl, allyl and butenyl).
The term xe2x80x9calkinylxe2x80x9d refers to a hydrocarbon chain as defined for alkyl having at least one olefinic triple bond (including for example propinyl, butin-(1)-yl, etc.
The term xe2x80x9calkoxyxe2x80x9d, alone or in combination, means an alkyl ether group in which the term xe2x80x98alkylxe2x80x99 has the significance given earlier, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec.butoxy, tert.butoxy and the like.
The term xe2x80x9calkoxycarbonylxe2x80x9d refers to a group of the formula xe2x80x94C(O)Rc wherein Rc is alkoxy as defined above.
The term xe2x80x9chydroxyxe2x80x9d refers to the group xe2x80x94OH, the term xe2x80x9ccyanoxe2x80x9d to the group xe2x80x94CN.
The term xe2x80x9chydroxyalkylxe2x80x9d means an alkyl group as defined above which is substituted by a hydroxy group.
The term xe2x80x9cthioalkylxe2x80x9d and xe2x80x9ccyanoalkylxe2x80x9d refer to an alkyl group as defined above which is substituted by a xe2x80x94SH group or an xe2x80x94CN group, respectively.
The term xe2x80x9chalogenalkylxe2x80x9d refers to an alkyl group as defined above which is substituted by one to three halogen atoms, preferably fluoro, e.g. trifluoromethyl, 2,2,2-trifluoroethyl, etc.
The term xe2x80x9calkylthioalkylxe2x80x9d is a group of the formula alkyl-S-alkyl.
xe2x80x9cCarboxyalkylxe2x80x9d means an alkyl as defined above which is substituted by a HOOC-group.
The term xe2x80x9calkylcarbonylxe2x80x9d, alone or in combination, means an acyl group derived from an alkanecarboxylic acid, i.e. alkyl-C(O)xe2x80x94, such as acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl etc.
The term xe2x80x9ccycloalkylxe2x80x9d signifies a saturated, cyclic hydrocarbon group with 3-8, preferably 3-6 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and the like.
The term xe2x80x9caminoxe2x80x9d refers to the group xe2x80x94NH2.
The term xe2x80x9carylxe2x80x9d for R2xe2x80x94alone or in combinationxe2x80x94, refers to an aromatic carbocyclic radical, i.e. a 6 or 10 membered aromatic or partially aromatic ring, e.g. phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl or naphthyl, and most preferably phenyl. The aryl moiety is optionally substituted with one or more groups independently selected from halogen, preferably fluoro, alkoxycarbonyl, e.g. methylcarbonyl, carboxy, cyano, alkyl, alkoxy, phenyl, phenoxy, trifluormethyl, trifluormethoxy, 1,3-dioxolyl, or 1,4-dioxolyl, more preferably fluor, alkoxycarbonyl, alkyl, trifluoromethyl and trifluoromethoxy and most preferably fluor. The most preferred aromatic groups are 2,5-difluorobenzyl and 2,4,5-trifluorobenzyl.
The term xe2x80x9carylxe2x80x9d for R3xe2x80x94alone or in combinationxe2x80x94, refers to an aromatic carbocyclic radical, i.e. a 6 or 10 membered aromatic or partially aromatic ring, e.g. phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl or naphthyl, and most preferably phenyl. The aryl moiety is optionally substituted with one or more groups independently selected from halogen, alkoxycarbonyl, e.g. methylcarbonyl, carboxy, cyano, alkyl, alkoxy, phenyl, phenoxy, trifluormethyl, trifluormethoxy, 1,3-dioxolyl, or 1,4-dioxolyl, cyclohexyl, hydroxy, alkylamido, e.g. acetamido, nitro, alkylsulfonyl, e.g. methylsulfonyl, more preferably fluoro, chloro, bromo, alkoxy, carboxy, 1,4-dioxolyl, alkoxycarbonyl. The most preferred aromatic groups are phenyl, 4-fluorobenzyl, 4-carboxybenzyl, 2,3-dihydrobenzo[1,4]dioxihyl, 2-bromophenyl, 2-fluorophenyl, 2-methoxycarbonylphenyl, naphthyl and 4-methoxyphenyl.
The term xe2x80x9carylxe2x80x9d for R4 to R10xe2x80x94alone or in combinationxe2x80x94refers to an aromatic carbocyclic radical, i.e. a 6 or 10 membered aromatic or partially aromatic ring, e.g. phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl or naphthyl, and most preferably phenyl. The aryl moiety is optionally substituted with one or more groups independently selected from halogen, preferably fluor, alkoxycarbonyl, e.g. methylcarbonyl, carboxy, cyano, alkyl, alkoxy, phenyl, phenoxy, trifluormethyl, trifluormethoxy, hydroxy, alkylamido, e.g. acetamido, nitro, alkylsulfonyl, e.g. methylsulfonyl, more preferably alkyl or alkoxy.
The term xe2x80x9caryloxyxe2x80x9d refers to an aryl group as defined above attached to a parent structure via an oxy radical, i.e., aryl-Oxe2x80x94.
The term xe2x80x9cheteroarylxe2x80x9d for R2 and R4 to R10xe2x80x94alone or in combinationxe2x80x94refers to an aromatic mono- or bicyclic radical having 5 to 10, preferably 5 to 6 ring atoms, containing one to three heteroatoms, preferably one heteroatom, e.g. independently selected from nitrogen, oxygen or sulfur. Examples of heteroaryl groups are thiophenyl, isoxazolyl, thiazolyl, pyridinyl, pyrrolyl, imidazolyl, tetrazolyl, preferably pyridinyl, isoxazolyl or thiazolyl. Optionally, the heteroaryl group can be mono-, di- or tri-substituted, independently, with phenyl, alkyl, alkylcarbonyl, alkoxycarbonyl, hydroxy, amino, alkylamino, dialkylamino, carboxy, alkoxycarbonylalkyl, preferably alkyl.
The term xe2x80x9cheteroarylxe2x80x9d for R3xe2x80x94alone or in combinationxe2x80x94refers to an aromatic mono- or bicyclic radical having 5 to 10, preferably 5 to 6 ring atoms, containing one to three heteroatoms, preferably one heteroatom, e.g. independently selected from nitrogen, oxygen or sulfur. Examples of heteroaryl groups are pyridinyl, thiophenyl, isoxyzolyl, isoquinolyl, quinolyl, and 1H-benzo[d][1,3]oxazin-2,4-dione and indolyl, pyrimidine, pyridazine, and pyrazine, preferably pyridinyl, thiophenyl, isoxazolyl, isoquinolyl, quinolyl, and 1H-benzo[d][1,3]oxazin-2,4-dione and indolyl. Optionally, the heteroaryl group can be mono-, di- or tri-substituted, independently, with phenyl, alkyl, alkylcarbonyl, alkoxycarbonyl, hydroxy, amino, alkylamino, dialkylamino, carboxy, oxo, alkoxycarbonylalkyl, preferably alkyl.
The term xe2x80x9cheterocyclylxe2x80x9dxe2x80x94alone or in combinationxe2x80x94refers to a non-aromatic mono- or bicyclic radical having 5 to 10, preferably 5 to 6 ring atoms, containing one to three heteroatoms, preferably one heteroatom, e.g. independently selected from nitrogen, oxygen or sulfur. Optionally the heterocyclic ring can be substituted by a group independently selected from halogen, alkyl, alkoxy, oxocarboxy, alkoxycarbonyl, etc. and/or on a secondary nitrogen atom (i.e. xe2x80x94NHxe2x80x94) by alkyl, arylalkoxycarbonyl, alkylcarbonyl or on a tertiary nitrogen atom (i.e. xe2x95x90Nxe2x80x94) by oxido. Examples for heterocyclic groups are morpholinyl, pyrrolidinyl, piperidyl, etc., and especially for R2 alkyl-pyran-triol-yl.
The term xe2x80x9cdimeric formxe2x80x9d means a compound wherein the two R1 groups of two identical compounds of formula I have been replaced by a common single bond or wherein R1 is glutathione-Sxe2x80x94 or cysteine-Sxe2x80x94 or ester and/or alkylcarbonyl or arylcarbonyl derivatives thereof, e.g. acetylcysteine-Sxe2x80x94 or benzoylcysteine-Sxe2x80x94, preferably glutathione-Sxe2x80x94, cysteine-Sxe2x80x94, acetylcysteine-Sxe2x80x94 or benzoylcysteine-Sxe2x80x94.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d refers to those salts that retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. The salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxylic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcystein and the like. In addition these salts may be prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium salts and the like. Salts derived from organic bases include, but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polymine resins and the like.
xe2x80x9cPharmaceutically acceptable estersxe2x80x9d means that compounds of general formula (I) may be derivatised at functional groups to provide derivatives which are capable of conversion back to the parent compounds in vivo. Examples of such compounds include physiologically acceptable and metabolically labile ester derivatives, such as methoxymethyl esters, methylthiomethyl esters and pivaloyloxymethyl esters. Additionally, any physiologically acceptable equivalents of the compounds of general formula (I), similar to the metabolically labile esters, which are capable of producing the parent compounds of general formula (I) in vivo, are within the scope of this invention.
The compounds of formula (I) are useful in inhibiting mammalian metalloprotease activity, particularly zinc hydrolase activity. More specifically, the compounds of formula (I) are useful as medicaments for the treatment and prophylaxis of disorders which are associated with diseases caused by endothelin-converting enzyme (ECE) activity. Inhibiting of this enzyme would be useful for treating myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma. In addition the compounds are useful as cytostatic and cerebroprotective agents for inhibition of graft rejection, for organ protection and for treatment of ophthalmological diseases.
In a preferred embodiment, the present invention comprises compounds of formula (I) wherein m and p are 0, n, o and q are 1. More specifically, the present invention comprises the above defined compounds of general formula (III). 
wherein R1, R2, R3, R4, X and Y are as defined as above.
In a preferred embodiment of the present invention, R1 is hydrogen or alkylcarbonyl, preferably hydrogen or acetyl, and more preferably hydrogen.
In a further preferred embodiment of the present R2 is aryl, arylalkyl, arylalkoxyalkyl, arylcarbamoyl, arylamino, arylcarbonyl, arylsulfonyl, cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl or heteroarylalkyl, more preferably aryl, arylalkyl, arylcarbamoyl, arylamino, arylcarbonyl, arylsulfonyl or heteroarylalkyl. In the most preferred R2 is arylalkyl and specifically phenylalkyl optionally substituted with 2 to 3 halogen atoms, e.g. 2,4,5-trifluoro-benzyl or 2,5-difluoro-benzyl.
According to the present invention R3 is preferably alkyl, halogenalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, cycloalkyl, halogenalkyl, alkoxyalkyl, alkoxycarbonylalkyl, alkinyl, aryl, arylalkyl, arylalkyl(alkoxycarbonyl)alkyl, arylcarbonylalkyl, aryloxyalkyl, arylalkenyl, aryl(alkoxycarbonyl)alkyl, heteroaryl, heteroarylalkyl or heterocyclyl, more preferably alkyl, arylalkyl, arylcarbonylalkyl, aryloxylakyl, alkylcycloalkyl, alkylcycloylkylalkyl, cycloalkyl, heteroarylalkyl or halogenalkyl and most preferably alkyl, arylalkyl, aryl, aryloxyalkyl or halogenalkyl, e.g. phenoxy-ethyl, 2,2,2-trifluoro-ethyl, 4-fluoro-benzyl, 4-carboxy-benzyl, 2,3-dihydrobenzo[1,4]dioxin-5-yl, 2-bromophenyl, butane-1-yl, methyl, benzyl, tert-butyl, 2-fluoro-phenyl, 4-fluoro-phenyl, 2-methoxy-carbonylphenyl, isopropyl, naphthalen-2-yl, naphthalen-2-yl, or 4-methoxy-phenyl.
In a preferred embodiment R4 is hydrogen.
In the above-defined compounds X is preferably xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2xe2x80x94NHxe2x80x94, xe2x80x94C(O)NR5xe2x80x94 or C(O)Oxe2x80x94, and more preferably xe2x80x94S(O)2xe2x80x94, xe2x80x94C(O)NHxe2x80x94 or C(O)Oxe2x80x94.
The invention comprises compounds as defined above, wherein R5 is hydrogen, alkyl or carboxyalkyl, preferably hydrogen.
R6 in the compounds described above is preferably hydrogen, alkyl or arylalkyl and more preferably hydrogen.
In further preferred embodiments of the present invention R7 is hydrogen or aryl and R8 is hydroxy or alkoxy.
In the present invention Y preferably is xe2x80x94Oxe2x80x94 or xe2x80x94NHxe2x80x94.
More specifically the invention comprises the above compounds wherein R1 is hydrogen or alkylcarbonyl, R2 is phenylalkyl substituted with 2 to 3 halogen; R3 is alkyl, aryl, arylalkyl, aryloxyalkyl or halogenalkyl, e.g. e.g. phenoxy-ethyl, 2,2,2-trifluoro-ethyl, 4-fluoro-benzyl, 4-carboxy-benzyl, 2,3-dihydrobenzo[1,4]dioxin-5-yl, 2-bromophenyl, butane-1-yl, methyl, benzyl, tert-butyl, 2-fluoro-phenyl, 4-fluoro-phenyl, 2-methoxy-carbonylphenyl, isopropyl, naphthalen-2-yl, naphthalen-2-yl, or 4-methoxy-phenyl;
X is xe2x80x94SO2xe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94; and Y is xe2x80x94NHxe2x80x94 or xe2x80x94Oxe2x80x94.
The present invention comprises compounds as defined above with the stereochemistry shown in formula (IV) 
wherein R1, R2, R3, R4, X and Y are as defined above.
In the most preferred embodiment the invention comprises compounds of formula (IV) wherein R1 is hydrogen or acetyl and R2 is difluorobenzyl or trifluorobenzyl, e.g. 2,4,5-trifluoro-benzyl or 2,5-difluoro-benzyl- and R3 is phenoxy-ethyl, 2,2,2-trifluoro-ethyl, 4-fluoro-benzyl, 4-carboxy-benzyl, 2,3-dihydrobenzo[1,4]dioxin-5-yl, 2-bromophenyl, butane-1-yl, methyl, benzyl, tert-butyl, 2-fluoro-phenyl, 4-fluoro-phenyl, 2-methoxy-carbonylphenyl, isopropyl, naphthalen-2-yl, naphthalen-2-yl, or 4-methoxy-phenyl and R4 is hydrogen, X is xe2x80x94S(O)2xe2x80x94, xe2x80x94C(O)NHxe2x80x94 or C(O)Oxe2x80x94 and Y is xe2x80x94Oxe2x80x94 or xe2x80x94NHxe2x80x94.
Preferred embodiments of the present invention are the compounds exemplified in the examples. Especially, the invention comprises the following compounds selected from the group consisting of
a) (3R,5S)-5-[(2,5-Difluoro-benzylamino)-methyl]-1-(naphthalene-2-sulfonyl)pyrrolidine-3-thiol;
b) (2S,4R)-2-[(2,5-Difluoro-benzylamino)-methyl]-4-mercapto-pyrrolidine-1-carboxylic acid (2-fluoro-phenyl)-amide;
c) (2S,4R)-2-[(2,5-Difluoro-benzylamino)-methyl]-4-mercapto-pyrrolidine-1-carboxylic acid 4-methoxy-phenyl ester;
d) (2S,4R)-2-[(2,5-Difluoro-benzylamino)-methyl]-4-mercapto-pyrrolidine-1-carboxylic acid 4-fluoro-phenyl ester;
e) 2S,4R)-2-[(2,5-Difluoro-benzylamino)-methyl]-4-mercapto-pyrrolidine-1-carboxylic acid isopropyl ester;
f) (2S,4R)-2-[(2,5-Difluoro-benzylamino)-methyl]-4-mercapto-pyrrolidine-1-carboxylic acid naphthalen-2-yl ester;
g) (2S,4R)-2-[(2,5-Difluoro-benzylamino)-methyl]-4-mercapto-pyrrolidine-1-carboxylic acid 2,3-dihydro-benzo [1,4]dioxin-5-yl ester;
h) (2S,4R)-2-[(2,5-Difluoro-benzylamino)-methyl]-4-mercapto-pyrrolidine-1-carboxylic acid butyl ester;
i) (2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-carboxylic acid isopropyl ester;
j) (2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-carboxylic acid 2,3-dihydro-benzo[1,4]dioxin-5-yl ester;
k) (2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-carboxylic acid tert-butyl ester;
l) (2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-carboxylic acid butyl ester;
m) (2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-carboxylic acid 2-fluoro-phenyl ester;
n) (2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-carboxylic acid 2-methoxycarbonyl-phenyl ester;
o) (2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-carboxylic acid 2-bromo-phenyl ester;
p) (3R,5S)-1-(Butane-1-sulfonyl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
q) (3R,5S)-1-Methanesulfonyl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol;
r) (2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-sulfonic acid benzylamide;
s) 4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-sulfonic acid butylamide;
t) 4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-sulfonic acid (2-phenoxy-ethyl)-amide;
u) 4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-sulfonic acid (2,2,2-trifluoro-ethyl)-amide;
v) 4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-sulfonic acid 4-fluoro-benzylamide;
w) 4-{[4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-sulfonylamino]-methyl}-benzoic acid;
x) (2S,4R)-4-Acetylsulfanyl-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-carboxylic acid 2,3-dihydro-benzo [1,4]dioxin-5-yl ester;
y) (2S,4R)-4-Acetylsulfanyl-2-[(2,5-difluoro-benzylamino)-methyl]-pyrrolidine-1-carboxylic acid butyl ester; and
z) (2S,4R)-4-Acetylsulfanyl-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-1-carboxylic acid 2-methoxycarbonyl-phenyl ester.
These compounds show activity values of 0.5 nM to 100 nM in the radioimmunoassay (E and F), see below.
The invention also refers to pharmaceutical compositions containing a compound as defined above and a pharmaceutically acceptable excipient.
A further embodiment of the present invention refers to the use of compounds as defined above as active ingredients in the manufacture of medicaments comprising a compound as defined above for the prophylaxis and treatment of disorders which are caused by endothelin-converting enzyme (ECE) activity especially myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, graft rejection, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection.
Further the invention refers to the use of compounds as described above for the treatment or prophylaxis of diseases which are associated with myocardial ischaemia, congestive head failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, graft rejection, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection.
In addition the invention comprises compounds as described above for use as therapeutic active substances, in particular in context with diseases which are associated with zinc hydrolase activity such as myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, graft rejection, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection.
The invention also comprises a method for the therapeutic and/or prophylactic treatment of myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, graft rejection, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection, which method comprises administering a compound as defined above to a human being or animal.
The invention also relates to the use of compounds as defined above for the inhibition of zinc hydrolase activity.
The invention also refers to the above compounds whenever manufactured by a process as described below.
Compounds of formula (I) can be prepared by methods known in the art or as described below. Unless otherwise indicated, the substituents R1, R2, R3, R4, R5, R6, R7, R8, X, Y, m, p, n, q are as described above.
Step a) of scheme I describes the persilylation of hydroxy- and amino groups, e.g. by reaction of compound 1 with hexamethyldisilazan/140xc2x0 C. followed by reaction with R3SO2Cl in THF or di-t-butyldicarbonate/NaHCO3 in dioxane/H2O (BOC protection). For inversion of the configuration (via mesylate) the resulting alcohol 2 is treated with MeSO3H/Ph3P/DIAD in toluene (room temperature to 80xc2x0 C.) or (via bromide) with LiBr/DEAD/Ph3P in THF (4xc2x0 C. to room temperature) or (via chloride) with Ph3P/CCl4 in CH2Cl2 (3xc2x0 C. to room temperature). In case of retention of the configuration (via mesylate) alcohol 2 can be transformed to a compound of formula 3 by reaction with MeSO2Cl/pyridine/DMAP (0xc2x0 C. to room temperature).
For the introduction of a protected thio moiety, e.g. triphenylmethanethiol or 4-methoxy-benzylmercaptane, compound of formula 3 are treated with Kxe2x80x94Otxe2x80x94Bu in DMF (for Br: 0xc2x0 C. to room temperature; for Cl: 0xc2x0 C.; for Mes: room temperature to 100xc2x0 C. For step d the corresponding compounds of formula 4, 8 and 9 can be obtained according to methods known in the art, e.g. LAH in THF at xe2x88x9220xc2x0 C. or Red-Al in Toluene/THF at xe2x88x9250xc2x0 C. 
For the reaction of compound 6 to compound 7 the Arndt-Eistert reaction may be used (in case q=2: hydrolysis with NaOH in EtOH at room temperature followed by addition of (COCl)2, cat DMF in CH2Cl2 at 0xc2x0 C. to room temperature to give the corresponding acid chloride followed by reaction with trimethylsilyldiazomethane in THF/CH3CN at 0xc2x0 C. to room temperature to give the corresponding diazoethanone and rearrangement to the methylester with silver benzoate in MeOH/THF at xe2x88x9225xc2x0 C. to room temperature gave 7). For q=3 compounds of formula 6 are hydrolyzed (NaOH in EtOH at room temperature), followed by formation of the corresponding Weinreb amide (e.g. HCl.H2NOMe/NMM, EDCI, HOBT) and conversion to an aldehyde (LAH, xe2x88x9278 to xe2x88x9230xc2x0 C. in THF). The obtained compound can be converted by a Horner-Emmons reaction (e.g. (EtO)2P(xe2x95x90O)CH2COOEt, NaH in THF) followed by reduction of the double bond and reduction of the ester (a) Mg in MeOH, (b) LAH in THF at xe2x88x9220xc2x0 C.) and BOC replacement (e.g. (a) TFA in CH2Cl2xe2x88x9220xc2x0 C. to room temperature, (b) NaHCO3/EtOAc, (c) ClCOOR3/Et3N or conversion to all other R3X described later).
For the introduction of Yxe2x95x90NR2, SR2 or a N-heterocycle (step g) a compound of formula 8 or 9 may be mesylated (1.1 eq MeSO2 Cl/1.5 pyridine/1 eq DMAP; in case Y is an amine the reaction is performed with e.g. 1 eq NaI, amine neat 100xc2x0 C., for Y R2 is pyrrole, imidazole or X is S, the reaction is performed with 1 eq NaI, NaH in DMF at 0xc2x0 C. to room temperature, followed by thiol deprotection, e.g. by treatment with TFA/Et3SiH, 0xc2x0 C. to room temperature (R1 is Trt) or TFA/Et3SiH, 0 to 80xc2x0 C. (R1 is PMB). An alternative method for the introduction of Yxe2x95x90NR2 would comprise the reaction of compound 8 or 9 with 1.1 eq MeSO2Cl/1.5 eq pyridine/1 eq DMAP (mesylation) followed by treatment with NaN3, DMF for 16 hours at 80xc2x0 C. to obtain the azide. This compound is then converted to the Yxe2x95x90NR2 (=amines or triazoles) after the introduction of new R3X.
For the introduction of a new R3X in case R3X is BOC, the azide may be BOC deprotected by reaction with TFA, CH2Cl2 at xe2x88x9220xc2x0 C. to room temperature, followed by reaction with ClCO2R3, iPr2NEt, CH2Cl2 or R3NCO in THF at 0xc2x0 C. to room temperature (or conversion to all other R3X described later), followed by reduction of the azide (e.g. Ph3P, THF, H2O or NaBH4, MeOH), followed by reductive amination (e.g. aldehyde, SnCl2, NaBH3CN, MeOH). In case R6 has to be introduced the compound is treated with R6Br/K2CO3 in acetonitrile at room temperature followed by thiol deprotection (e.g. Et3SiH, TFA, 0xc2x0 C. to room temperature or Et3SiH, TFA, MeCN at room temperature or, for selective trityl-thiol deprotection in the presence of BOC by for example treatment with iPr3SiH in TFA/CH2Cl2, 0xc2x0 C. to room temperature).
A further method for the introduction of new substituents YR2 and XR3 comprises the oxidation of an alcohol 8 or 9 to the aldehyde (e.g. with (COCl)2/DMSO/iPr2NEt at xe2x88x9265xc2x0 C. to room temperature in CH2Cl2), an imine formation (e.g. corresponding primary amine/MgSO4, room temperature, 16 hours in CH2Cl2), reduction to the amine (e.g. NaBH4 in MeOH at 40xc2x0 C., FMOC-protection of Yxe2x95x90NHR2 (e.g. FMOC-Cl/iPr2NEt/cat DMAP, 0xc2x0 C. to room temperature), BOC-deprotection (e.g. TFA in CH2Cl2, 0xc2x0 C. to room temperature), followed by reaction with R3NHSO2Cl (in e.g. iPr2NEt, 0xc2x0 C. to room temperature) (or conversion to all other R3X described later), FMOC-deprotection (e.g. Et2NH in THF), and thiol deprotection (e.g. Et3SiH in TFA at 80xc2x0 C.).
Compounds wherein YR2 is triazol may be prepared via step g by reaction of the above mentioned azide with the corresponding alkyl/amineCOCH2 keton/ester/amide/aryl and K2CO3 (in DMSO, 40xc2x0 C. for 3 days) followed by thiol deprotection (e.g. Et3SiH, TFA, 0xc2x0 C. to room temperature or Et3SiH, TFA, MeCN, room temperature). An alternative is the reaction of the corresponding azide with alkyl/amineCOCH2 ester, K2CO3, DMSO, 40xc2x0 C. for 3 days, followed by hydrolysis of the ester(e.g. LiOH, THF) and thiol deprotection as described above.
In case of an introduction of a new substituent R3X in case R3X is BOC, the corresponding compound may be prepared via step g by BOC deprotection (TFA, CH2Cl2, xe2x88x9220xc2x0 C. to room temperature), followed by reaction with a compound of formula R3OCOCl and iPr2NEt/CH2Cl2 or conversion to all other R3X described later.
In case YR2 represents a phenolether the phenol may be introduced via step g under Mitsunobu conditions (e.g. DEAD/Ph3P/PhOH in THF) and in case R1 is Trt followed by reaction with e.g. TFA/Et3SiH at 0xc2x0 C. to room temperature or, in case R1 is PMB, followed by reaction with e.g. TFA/Et3SiH, at 0 to 80xc2x0 C. In case YR2 represents carbamates, the corresponding compounds 5 may by obtained via step g by reaction with isocyanate/NMM in toluene at room temperature followed optionally by reaction with the corresponding alkyl-, cycloalkyl-halogenide, alkylbromoacetate with NaH in DMF at 0xc2x0 C. to room temperature. If YR2 represents an ether the corresponding compounds 5 may be obtained via step g by O-alkylation (e.g. NaH, R2-halogenide, DMF 0xc2x0 C. to room temperature) or by O-alkylation with phase transfer conditions (e.g. R2-halogenide/50% NaOH, Bu4NHSO4). This reaction may be followed by reaction with e.g. TFA/Et3SiH at 0xc2x0 C. to room temperature (R1 is Trt) or, in case R3 is PMB, followed by reaction with e.g. TFA/Et3SiH, at 0 to 80xc2x0 C.
Compounds containing a group of formula (II) may be prepared via step g by reaction of the corresponding starting compound 8 or 9 with NaH, R2-halogenide/NaI, DMF and in case R2 contains a COOtBu (a) reaction with TFA in CH2Cl2 at xe2x88x9220xc2x0 C. and (b) reaction with EDCI/HOBT amine in CH2Cl2 for formation of the corresponding amidexe2x80x94or, in case R2 contains a COO-alkyl- (a) reaction with 1N NaOH in THF/EtOH to give the acid. Both pathways are completed by reaction with Et3SiH in TFA at 0xc2x0 C. to room temperature.
Compounds wherein YR2 is an ether the group R3X may be varied as followed: For O-alkylation compounds of formula 8 or 9 may be reacted with e.g. NaH/reactive R2Br in DMF at 0xc2x0 C. to room temperature followed be BOC deprotection (e.g. TFA in CH2Cl2 at xe2x88x9220xc2x0 C. to room temperature to get the amine as starting material.
In case R3X is a carbamate these starting compounds may be reacted with R3OCOCl/pyridine in THF or by reaction with (a) R3OH/Cl3COCl/quinoline (formation of the chloroformate) followed by reaction with NaH. In case R3X is a sulfonamide the starting compounds may be reacted with R3SO2Cl/(i-Pr)2EtN/cat DMAP in ClCH2CH2Cl at room temperature. In case R3X is urea the starting compounds may be reacted with isocyanate in EtOH at room temperature. In case R3X is an alkylated urea, (i.e. introduction of R5) the starting compounds may be reacted with isocyanate in EtOH at room temperature followed by reaction with the corresponding alkylhalogenide/Kxe2x80x94OtBu at 0xc2x0 C. to room temperature. In case R3X is an amide, the starting compounds may be reacted with RCOOH/EDCI/DMAP (with anhydride formation, and subsequent addition of the starting amine, xe2x88x9210xc2x0 C. to room temperature) or as alternative with RCOOH/EDCI/DMAP at room temperature. In case R3X is a sulfamide (for R3 is NH2) the starting compounds may be reacted with sulfamic acid 2,4,6-trichlorophenylester/Et3N in CH2Cl2 at 40xc2x0 C. or with other methods which are known in the art. In case R3X is SO2OH the starting compounds may be reacted with chlorosuphonic acid/2-picoline. In case R3X is an alkylated sulfamide (i.e. introduction of R5) the starting compounds may be reacted with NaH/alkyl halide in DMF at 0xc2x0 C. at room temperature. Thiol liberation can than be achieved by reaction in TFA/Et3SiH at room temperature.
Step h of scheme 1 includes a reaction pathway for the preparation of further derivatives by reaction of compounds of formula 4 with (a) NaH/reactive R2Br in DMF at 0xc2x0 C. to room temperature followed by (b) reaction with KSAc in DMF at 100xc2x0 C. The corresponding thiols could be obtained by reaction of the the above compounds with LiOH aqueous in EtOH at 0xc2x0 C. to room temperature.
Step i of scheme 1 shows the preparation of compounds of formula 5 wherein Y is C. Compounds of formula 4 are treated with NaOH in EtOH at room temperature, followed by formation of a Weinreb amide (e.g. by reaction with HCl.H2NOMe/NMM, EDCI, HOBT at 0xc2x0 C. to room temperature), followed by formation of the corresponding ketone (e.g. by reaction with R2xe2x80x94MgBr in THF, at 0xc2x0 C. to room temperature), BOC deprotection (e.g. TFA in CH2Cl2, at xe2x88x9220xc2x0 C. to room temperature) followed by reaction with R3OCOCl/NMM in CH2Cl2 (or conversion to all other R3X described before) and reduction of the ketone to the methylene and thiol deprotection (e.g. Et3SiH in TFA at 80xc2x0 C. for 18 hours). 
Further reaction pathways are shown in scheme 2: Compounds of formula 2 may be obtained by persilylation of the hydroxy- and amino groups of compounds 1 (e.g. reaction with HMDS, neat 120xc2x0 C.) and preparation of the corresponding methyl ester (step a) (e.g. R3SO2Cl, iPr2NEt, THF, if acid, then e.g. MeOH, HCl). Step b of scheme 2 shows the formation of the corresponding t-Butyldimethylsilylether or for example the t-butylether (e.g. by reaction with TBDMSCI/DBU in CH3CN at 0xc2x0 C. to room temperature, or by reaction with isobutylene, BF3.OEt2 in CH2Cl2 at xe2x88x9230 to xe2x88x9220xc2x0 C. Step c comprises the reaction of compound 2 with LiBH4 in THF at xe2x88x9220xc2x0 C. to room temperature or LAH at xe2x88x9215xc2x0 C. in ether to obtain compounds 4.
In case R2Y is R2N, step d of scheme 2 shows the introduction of a phthalimide under Mitsunobu conditions (e.g. phthalimide, DEAD/Ph3P in THF, 3 to 80xc2x0 C. This may be followed by t-butyldimethylsilylether deprotection (e.g. for t-BuMe2Si: reaction with TBAF in THF at room temperature), followed by reaction with e.g. MeSO3H/DIAD/Ph3P in toluene at room temperature xe2x88x9280xc2x0 C., followed by e.g. reaction with KSCOCH3 in DMF at 100xc2x0 C., followed by phthalimide deprotection and disulfide formation (e.g. by reaction with CH3NH2, EtOH for 2 days at room temperature which may be followed by reaction with R2SO2Cl or R2COCl, DMAP in CH2Cl2 or R2CO2H, TPTU or N-alkylation by reaction with R2Br and N-methylmorpholine in CH2Cl2. This may be followed by side chain manipulation e.g. hydrolysis with LiOH in THF/H2O. Step e of scheme 2 shows the reduction of the disulfide to the thiol (e.g. by nBu3P/CF3CH2OH/H2O at 0xc2x0 C. or DTT, 2 M K2CO3, MeCN).
In case R2Y is R2O: Compounds of this type may be obtained by reaction shown in step f and g. These reaction may comprise (step f) reaction of compounds 4 with R2Br/NaH in DMSO at room temperature or benzyl-2,2,2-trichloroacetimidate/CF3SO3H in CH2Cl2/cyclohexane at room temperature (here the R2-side chains may be manipulated by reaction with 10% Pd/C/H2 in EtOH/dioxane) or the reaction is performed with PhOH/Ph3P/DIAD in THF at room temperature. All reactions may be followed by removal of the t-Bu-ether in TFA at 0xc2x0 C. to room temperature. For the preparation of compounds of formula 7 (step g) compounds 6 (if m=0: for inversion of the configuration) may be obtained by thioacetate formation under Mitsunobu conditions (e.g. CH3COSH/Ph3P/DIAD in THF at 0xc2x0 C. to room temperature) followed by formation of the thiol (e.g. by reaction with MeONa in MeOH at 0xc2x0 C.; plus potential side chain manipulation with 1 N Na2CO3 in MeOH) or (if m=0: for retention of configuration) reaction of compounds 6 for e.g. formation of the mesylate (MeSO3H/Et3N/Ph3P/DIAD in toluene at 0xc2x0 C. to 85xc2x0 C.) followed by preparation of the thioacetate (e.g. KSCOCH3 in DMF at 100xc2x0 C.) and formation of the thiol (MeONa in MeOH at 0xc2x0 C.).
Scheme 3 shows a further route for the preparation of compounds of formula (I). Step a comprises the preparation of compounds 2 by reaction of compounds 1 with e.g. R3SO2Cl/DMAP in CH2Cl2 at room temperature or Et3N in CH2Cl2 (reflux)) followed by monohydrolysis (e.g. 1 M NaOH in MeOH/H2O for 20 min reflux and reduction of the acid with e.g. BH3.THF in THF at 0xc2x0 C. Step c shows an alkylation (e.g. with R2Br/NaH in DMSO at room temperature) followed by ester reduction (e.g. LAH at xe2x88x9215xc2x0 C. in ether). Step e comprises the formation of the thioacetate (e.g. by CH3COSH/Ph3P/DIAD in THF at 0xc2x0 C. to room temperature followed by formation of the thiol (e.g. with MeONa in MeOH at 0xc2x0 C. Step f shows the reduction of both esters (e.g. with LAH in THF at 0xc2x0 C.) followed by monoalkylation (e.g. with R2Br/NaH in DMF at xe2x88x9215xc2x0 C., step g). This pathway may be continued by formation of the mesylate (e.g. with MeSO2Cl/Et3N in Et2O at xe2x88x9220xc2x0 C. to room temperature), formation of the thioacetate (e.g. with KSCOCH3 in DMF at 100xc2x0 C.) and formation of the thiol (LAH in Et2O reflux). Step h depicts an additional way for preparing a mono-p-tosylate (e.g. with p-TosCl/Et3N/cat DMAP in THF at room temperature) followed by introduction of the tritylthiolate (e.g. with Ph3CSH/KOtxe2x80x94Bu in DMF at room temperature), formation of the mesylate (step i, with MeSO2Cl/Et3N in THF at 0xc2x0 C. to room temperature) followed by the formation of the phenolether (e.g. with PhOH/NaH in DMF at room temperature) or alternatively alkylation of the alcohol directly with R2Br/NaH in DMF at xe2x88x9215xc2x0 C. to room temperature and finally formation of the thiol (e.g. with Et3SiH in TFA at 0xc2x0 C. to room temperature). 
Scheme 4 shows in step a the selective protection of the thiol group (e.g. by reaction with Ac2O/pyridine in CH2Cl2 at room temperature; the starting compound can be received by a deprotection of a STrt or SPMB protected alcohol described above, e.g. Et3SiH in TFA). The S-acetylated alcohol was then reacted with (a) 1,2,3,4-tetra-O-acetyl-t-deoxy-beta-L-mannospyranose/trimethylsilyl-trifluoromethanesulphonate in CH2Cl2 at 0xc2x0 C. followed by cleavage of all acetyl group with NaOMe in MeOH at 0xc2x0 C.
Scheme 5 shows the reaction pathway for the synthesis of sterically hindered thiols. Step a represents a Swern-oxidation of the starting material which is known in the art (e.g. (COCl)2/DMSO/Et(i-Pr)2N in CH2Cl2). Step b shows the methylene introduction by a Wittig reaction (e.g. with Ktxe2x80x94BuO/CH3PPh3Br in THF at room temperature to 70xc2x0 C. Step c shows a reduction via a mixed anhydride (e.g. with iBuOCOCl/NMM in THF at xe2x88x925xc2x0 C. to room temperature, then the mixture is added to NaBH4 in water at 0xc2x0 C. and warmed up to room temperature) followed by alkylation of the corresponding alcohol (e.g. with NaH/R2Br in THF at 0xc2x0 C. to room temperature). Step d represents the formation of an epoxide (e.g. with mCPBA in CH2Cl2 at room temperature) followed by the formation of a thiirane (e.g. with KSCN in EtOH/H2O at room temperature or PO(OMe)2SCl in CH2Cl2). The resulting diastereomers are separable with methods known in the art. Step e shows the opening of the thiirane (e.g. with LiHBEt3 in THF and LAH) and reduction of the resulting disulfide (e.g. with P(Bu)3/H2O in trifluoroethanol/CH2Cl2). 
Scheme 6 shows the synthesis of further derivatives: for Y being NH: Step a comprises the N-acylation (protection of NH, e.g. with AcCl, iPr2NEt, 4-(N-benzyl-N-methylamino)pyridine polymer-supported, CH2Cl2) or by N-CBz-Protection (e.g. with BnOCOCl, iPr2NEt, 4-Benzyl-N-methylamino)pyridine polymer supported, CH2Cl2), followed by selective BOC deprotection (e.g. with TFA, CH2Cl2 at xe2x88x9220xc2x0 C. to room temperature) and reaction with a reactive R3 derivative (e.g. R3CO2Cl, iPr2NEt, 4-(N-Benzyl-N-methylamino)pyridine polymer-supported, CH2Cl2; step c(or conversion to all other R3X described before)) and formation of the thiol (e.g. with iPr3SiH, TFA, CH2Cl2).
For Y being C-, protected-Nxe2x80x94, O- or S-substituents step e of scheme 6 shows formation of S-compounds of thiol inhibitors of formula (I) by (a) reaction of the free thiol with for example AcCl in pyridine or PhCOCl in pyridine at 0xc2x0 C. to room temperature or (b) a S-derivative synthesis (e.g. with BOC-Cys(Npys)-OH=2-(BOC-Cys)disulfanyl-3-nitro-pyridine or Ac-Cys(Npys)-OH=2-(acetyl-Cys)disulfanyl-3-nitro-pyridine) in DMF/0.1 M phosphate buffer (pH 6.2). The reaction for Y being protected N-atoms (Y deprotection) can be performed selective with for example HBr/AcOH in EtOAc. Step f shows the formation of the thiol as described above. 
The present invention also refers to the above described processes, especially to processes for the preparation of a compound of the present invention comprising reaction of a compound of formula V 
wherein R1, R3, R4, X, Y, m, n, o, q and p are as defined above and A is a HS-protecting group
a) with a R2-halogenide for introduction of a xe2x80x94OR2 group: or
b) mesylation of a compound of formula (V), followed by reaction with H R6Nxe2x80x94R2 or HSR2 or HN-heterocycles for introduction of a xe2x80x94NR6xe2x80x94R2 or xe2x80x94SR2 group or xe2x80x94N-heterocycle;
optionally followed by conversion of a R3xe2x80x94X group into a different oneand/or deprotection and/or thiol liberation.
Dimeric forms of a compound of formula I may be prepared by oxidative treatment of the formula I monomers.
On the basis of their capability of inhibiting metalloprotease activity, especially zinc hydrolase activity, the compounds of formula I can be used as medicaments for the treatment and prophylaxis of disorders which are associated with vasoconstriction of increasing occurrences. Examples of such disorders are high blood pressure, coronary disorders, cardiac insufficiency, renal and myocardial ischaemia, renal insufficiency, dialysis, cerebral ischaemia, cardiac infarct, migraine, subarachnoid haemorrhage, Raynaud syndrome and pulmonary high pressure. They can also be used in atherosclerosis, the prevention of restenosis after balloon-induced vascular dilation, inflammations, gastric and duodenal ulcers, ulcus cruris, gram-negative sepsis, shock, glomerulonephtritis, renal colic, glaucoma, asthma, in the therapy and prophylaxis of diabetic complications and complications in the administration of cyclosporin, as well as other disorders associated with endothelin activities.
The ability of the compounds of formula (I) to inhibit metalloprotease activity, particularly zinc hydrolase activity, may be demonstrated by a variety of in vitro and in vivo assays known to those of ordinary skill in the art. Pharmaceutically acceptable esters, pharmaceutically acceptable salts and dimeric forms of the compounds of formula I can also be tested by those of ordinary skill in the art for their ability to inhibit metalloprotease activity.
A) Cell Culture
A stable human umbilical vein endothelial cell line (ECV304) was cultured in xe2x80x9ccell factoriesxe2x80x9d as described until confluency (Schweizer et al. 1997, Biochem. J. 328: 871-878). At confluency cells were detached with a trypsin/EDTA solution and collected by low speed centrifugation. The cell pellet was washed once with phosphate buffered saline pH 7.0 and stored at xe2x88x9280xc2x0 C. until use.
B) Solubilization of ECE from ECV304 Cells
All procedures were performed at 0-4xc2x0 C. if not stated otherwise. The cell pellet of 1xc3x97109 cells was suspended in 50 ml of buffer A (20 mM Tris/HCl, pH 7.5 containing 5 mM MgCl2, 100 xcexcM PMSF, 20 xcexcM E64, 20 xcexcM leupeptin) and sonicated. The resulting cell homogenate was centrifuged at 100,000 gav for 60 minutes. The supernatant was discarded and the resulting membrane pellet was homogenized in 50 ml buffer A and centrifugated as described. The washing of the membrane fraction in buffer A was repeated twice. The final membrane preparation was homogenized in 50 ml of buffer B (buffer A+0.5% Tween 20 (v/v), 0.5% CHAPS (w/v), 0.5% Digitonin (w/v)) and stirred at 4xc2x0 C. for 2 hours. Thereafter the remaining membrane fragments were sedimented as described. The resulting clear supernatant containing the solubilized ECE was stored in 1.0 ml aliquots at xe2x88x92120xc2x0 C. until use.
C) ECE Assay
The assay measured the production of ET-1 from human big ET-1. To measure high numbers of samples an assay performed in 96 well plates was invented. The enzyme reaction and the radioimmunological detection of the produced ET-1 was performed in the same well, using a specifically developed and optimized coating technique.
D) Coating of Plates
Fluoronunc Maxisorp White (code 437796) 96 well plates were irradiated with 1 joule for 30 minutes in a UV Stratalinker 2400 (Stratagene). The 96 well plates were then fill with 300 xcexcl protein A solution (2 xcexcg/ml in 0.1 M Na2CO3 pH 9.5) per well and incubated for 48 hours at 4xc2x0 C. Coated plates can be stored for up to 3 weeks at 4xc2x0 C. until use.
Before use the protein A solution is discarded and the plates are blocked for 2 hours at 4xc2x0 C. with 0.5% BSA in 0.1M Na2CO3, pH 9.5.
Plates were washed with bidestilled water and were ready to perform the ECE assay.
E) Screening Assay
Test compounds are solved and diluted in DMSO. 10 xcexcl of DMSO was placed in the wells, followed by 125 xcexcl of assay buffer (50 mM Tris/HCl, pH 7.0, 1 xcexcM Thiorphan, 0.1% NaN3, 0.1% BSA) containing 200 ng big ET-1. The enzyme reaction was started by the addition of 50 xcexcl of solubilized ECE (diluted in assay buffer 1:30 to 1:60 fold (v/v)). The enzyme reaction was carried out for 30 minutes at 37xc2x0 C. The enzyme reaction was stopped by addition of 10 xcexcl 150 mM ETDA, pH 7.0.
F) Radioimmunoassay
The ET-1 RIA was performed principally as described earlier (Lxc3x6ffler, B.-M. and Maire, J.-P. 1994, Endothelium 1: 273-286). To plates containing the EDTA stopped enzyme reaction mixture 25 xcexcl of assay buffer containing 20000 cpm (3-(125I)Tyr)-endothelin-1 and 25 xcexcl of the ET specific antiserum AS-3 (dilution in assay buffer 1:1000) was added. Plates were incubated under mixing at 4xc2x0 C. over night. Thereafter, the liquid phase was sucked with a plate washer and plates were washed once with bidestilled water. To the washed plates 200 xcexcl scintillation cocktail (Microscint 40 LSC-Cocktail, Packard, code 6013641) was added and plates were counted for 2 minutes per well in a Topcount.
Standard curves were prepared in plates with synthetic ET-1 with final concentrations of 0 to 3000 pg ET-1 per well. In all plates controls for maximal ECE activity (in the presence of 10 xcexcl DMSO) and for background production of ET-1 immunoreactivity (in the presence of 10 mM EDTA or 100 xcexcM phosphoramidon) were performed. Assays were run in triplicate.
G) Kinetic Assay
The described assay format could be used to determine the kinetic characteristics of the used ECE preparation as well as different ECE inhibitors (i.e. Km, Ki) by variation of the substrate concentration used in the assay.
H) Cell Based ECE Assay
Human ECE-1c was stable expressed in MDCK cells as described (Schweizer et al. 1997, Biochem. J. 328: 871-878). Cells were cultured in 24 well plates to confluency in Dulbecco""s modified Eagles""s medium(DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 0.8 mg/ml geneticin, 100 i.u./ml penicillin and 100 xcexcg/ml streptomycin in a humidified air/CO2 (19:1) atmosphere. Before ECE assay the medium was replaced by 0.5 ml DMEM-HBSS 1:1, 10 mM HEPES pH 7.0 supplemented with 0.1% (w/v) BSA. The inhibitors were added in DMSO at a final concentration of 1%. The enzyme reaction was started by the addition of 0.42 xcexcM human big ET-1 and performed for 1.5 hours at 37xc2x0 C. in an incubator. At the end of incubation, the incubation medium was quickly removed and aliquots were analysed by radioimmunoassay for produced ET-1 as described above.
The ECE screening assay was validated by the measurement of the characteristic inhibitor constants of phosphoramidon (IC50 0.8xc2x10.2 xcexcM) and CGS 314447 (IC50 20xc2x14 nM) [De Lombaert, Stephane; Stamford, Lisa B.; Blanchard, Louis; Tan, Jenny; Hoyer, Denton; Diefenbacher, Clive G.; Wei, Dongchu; Wallace, Eli M.; Moskal, Michael A.; et al. Potent non-peptidic dual inhibitors of endothelin-converting enzyme and neutral endopeptidase 24.11. Bioorg. Med. Chem. Lett. (1997), 7(8), 1059-1064]. All three inhibitors were measured with IC50 values not significantly different from those described in the literature but measured with different assay protocols. In the cell based assay phosphoramidon showed an IC50 of 4 xcexcM. This assay gave additional information about the inhibitory potency of inhibitors under much more physiologic conditions, as e.g. the ECE was embedded in a normal plasma membrane environment. It is important to state, that the screening assay was performed in the presence of 1 xcexcM Thiorphan to block any potential big ET-1 degradation due to the action of NEP24.11. No NEP activity was present in MDCK-ECE-1c transfected cells in preliminary experiments when ET-1 production was measured in presence or absence of thiorphan. In subsequent experiments no thiorphan was added in the incubation medium.
According to the above methods, the compounds of the present invention show activity values in the radioimmunoassay (E and F) of about 0.5 nM to about 100 xcexcM. The preferred compounds show values of 0.5 nM to 100 nM.
As mentioned earlier, medicaments containing a compound of formula I are also an object of the present invention as is a process for the manufacture of such medicaments, which process comprises bringing one or more compounds of formula I and, if desired, one or more other therapeutically valuable substances into a galenical administration form.
The pharmaceutical compositions may be administered orally, for example in the form of tablets, coated tablets, drages, hard or soft gelatin capsules, solutions, emulsions or suspensions. Administration can also be carried out rectally, for example using suppositories; locally or percutaneously, for example using ointments, creams, gels or solutions; or parenterally, for example using injectable solutions.
For the preparation of tablets, coated tablets, dragees or hard gelatin capsules the compounds of the present invention may be admixed with pharmaceutically inert, inorganic or organic excipients. Examples of suitable excipients for tablets, dragees or hard gelatin capsules include lactose, maize starch or derivatives thereof, talc or stearic acid or salts thereof.
Suitable excipients for use with soft gelatin capsules include for example vegetable oils, waxes, fats, semi-solid or liquid polyols etc. According to the nature of the active ingredients, it may, however, be the case that no excipient is needed at all for soft gelatin capsules.
For the preparation of solutions and syrups, excipients which may be used include for example water, polyols, saccharose, invert sugar and glucose. For injectable solutions, excipients which may be used include for example water, alcohols, polyols, glycerin, and vegetable oils. For suppositories, and local or percutaneous application, excipients which may be used include for example natural or hardened oils, waxes, fats and semi-solid or liquid polyols.
The pharmaceutical compositions may also contain preserving agents, antioxidants, solubilising agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for the variation of osmotic pressure, buffers, coating agents or antioxidants. They may also contain other therapeutically valuable agents.
The dosages in which the compounds of formula I are administered in effective amounts depend on the nature of the specific active ingredient, the age and the requirements of the patient and the mode of application. In general, dosages of 0.1-100 mg/kg body weight per day come into consideration, although the upper limit quoted can be exceeded when this is shown to be indicated.
The following specific examples are provided as a guide to assist in the practice of the invention, and are not intended as a limitation on the scope of the invention.